The overall goal of this project is to understand the biochemical basis for the regulation of the inactivation of cocaine by its enzyme-mediated hydrolysis to inactive metabolites, and the regulation of formation of the active metabolite benzoylecgonine ethyl ester or ethylcocaine, which is formed from alcohol and cocaine. The duration and stimulant effects of cocaine are limited principally by its rate of hydrolysis to benzoylecgonine and ecgonine methyl ester. These major metabolites are hot pharmacologically active as psychomotor stimulants. Two different esterases have been identified in liver that catalyze the hydrolysis of cocaine to benzoylecgonine and ecgonine methyl ester, respectively. The studies in this application will focus on the purification of these two esterases by column chromatography and the characterization of their molecular properties, viz. molecular weight, subunit structure and partial amino acid sequence. The substrate and inhibitor specificity of these enzymes will be examined by steady-state and stopped flow kinetic analysis. An active metabolite of cocaine, ethylcocaine, has been identified in individuals taking alcohol and cocaine concurrently. In rat liver and brain, the ethylcocaine concentration reaches 18-30% of the cocaine level after injection of alcohol and cocaine. It has been shown that ethylcocaine is more potent than cocaine in mediating lethality, and that coabuse of cocaine and ethanol leads to increased hepatotoxicity as compared to cocaine alone. The liver esterase that hydrolyzes cocaine to benzoylecgonine will also catalyze the transesterification of cocaine to ethylcocaine in the presence of ethanol. The kinetics of ethylcocaine formation by. the purified esterase will be examined. Antibodies will be prepared to purified cocaine esterases and ethyltransferases, and their distribution in human tissues will be examined by immunoblotting. The reasons for the common coabuse of alcohol and cocaine, and the nature of the rewarding effects of this combination of drugs versus either drug alone is not known. Rats have now been shown to self-administer ethylcocaine intracranially to the medial prefrontal cortex, similar to that of cocaine. The reinforcing effects and potential abuse liability of ethylcocaine will be assessed by determining conditions under which rats will intravenously and intracranially selfadminister ethylcocaine compared with cocaine. The effects of ethanol on intravenous cocaine self-administration will also be examined. These studies should provide insight into the high incidence of the combined use of these drugs and potential health risk of this practice.